ABSTRACT Hematopoiesis is a highly regulated multistep process that requires intact DNA repair mechanisms to maintain a normal stem cell pool and prevent malignancy development. As part of my NHLBI funded K08, I demonstrated for the first time in vivo that mice lacking Brca1 in hematopoietic stem cells (HSCs) develop spontaneous bone marrow failure (BMF) and hematopoietic malignancies featuring numerous chromosomal translocations. However, the DNA repair pathway(s) that operate in HSCs in the absence of Brca1 to repair double strand breaks (DSBs) and, thus, contribute to these translocations as well as whether additional cooperating genomic events must occur to allow cell survival in the face of genomic instability in Brca1 deficient HSCs in vivo remain unknown. The central hypothesis of this R03 application is that Brca1 deficiency leads to accumulation of unrepaired DNA damage resulting either in sequential cell cycle arrest, apoptosis, and BMF or the utilization of error-prone DSB repair pathways that promote and select for additional genomic events to allow cell cycle progression and survival, ultimately leading to malignancy. I will test this hypothesis with the following aims: Aim 1: Determine the major DNA repair pathway(s) utilized in proliferating Brca1-deficient HSCs. To do this, I will isolate HSCs from Brca1+/+ and Brca1-/- murine bone marrow at baseline and post proliferation inducing hematopoietic stressors that are known to increase replication related DSBs in wild-type HSCs. I will determine and compare the proportions of HSCs with DNA damage events, the major pathway(s) recruited to repair or tolerate this damage, and whether HSCs with damage can progress through the cell cycle. Aim 2: Determine the sequence of genomic events that occur pre- & post-Brca1 deletion that cooperate to cause BMF and malignant transformation. To do this, I will perform whole genome and RNA sequencing on HSCs isolated from Brca1fl/fl mice before and at multiple time points after Brca1 loss, including at the time of spontaneous BMF and hematologic malignancy development in vivo, to determine the stepwise sequence of events that cooperate with Brca1 deficiency to cause overt disease. Successful completion of these R03 aims will inform a detailed, evidence-based model of Brca1-mediated BMF and cancer development that I will then test in both murine and human samples in a future R01 application. The work outlined in this proposal will serve as additional critical training in techniques to interrogate DNA repair pathway fidelity and to detect specific DNA repair deficits in vivo, key skill sets that will accelerate my career path toward independence and allow me to pursue future clinical assays to detect and drugs to target unique DNA repair dependences in various marrow failure conditions and cancers.